Ligation Independent Cloning

Ligation Independent Cloning employs the 3′-5′ exonuclease activity of T4 DNA polymerase in order to create 5′ overhangs on both the vector and insert. In the presence of a single free dNTP, T4 polymerase will continue to function as an exonuclease until a base is exposed on the single strand overhang which is complementary to the free nucleotide. Given this opportunity, T4 will resume its polymerase activity, add back the free base, and become stuck at this point (with no other free bases to add). Complementary overhangs are built into the PCR primers for the insert, based on the destination vector sequence and choice of restriction site. Because of the relatively long stretches of base pairing in the annealed product, ligation is rendered unnecessary. The product may be transformed directly into E. coli, where the nicks will be repaired by the normal replication process. The downside of restriction enzyme cloning is that you can hardly assemble more than 3 fragments simultaneously, which requires multiple cloning steps to built-up an entire plasmid. In addition, ligation independent cloning does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.


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